New fruit fly model narrows down toxic nature of C9orf72 mutation

A new study led by Packard scientist Fen-Biao Gao has provided surprising clues as to how the C9orf72 mutation harms motor neurons and leads to ALS.

Frontotemporal Dementia (FTD) is associated with ALS and several genes are involved in the pathogenesis including CHMP2B, TDP-43, FUS, and C9ORF72. How these mutant proteins cause or contribute to neuronal dysfunction and neurodegeneration in FTD remains poorly defined.

Fen Biao Gao, neurologist at University of Massachusetts Medical School has led this research.

A new study in the journal Neuron, led by Packard scientist Fen-Biao Gao, a neuroscientist at the University of Massachusetts Medical School (UMMS) in collaboration with Zhiping Weng of UMMS and Leonard Petrucelli of the Mayo Clinic, has provided surprising clues as to how the C9orf72 mutation harms motor neurons and leads to ALS. Because many neurons in ALS had clumps of RNA in the nucleus, researchers believed that these were causing damage. But Gao’s recent work, using a fruit fly model of ALS, shows that this may not be the case. Instead, Gao hypothesizes that toxicity occurs when these RNAs escape into the cytosol and are translated.

Four years ago, an international team of researchers, including Packard scientists, showed that a series of six nucleotides were repeated hundreds, even thousands, of times in chromosome 9 in up to 45 percent of people with familial ALS. They had discovered the C9orf72 mutation, the most common genetic cause of ALS and frontotemporal dementia (FTD), but they had no idea what the gene did or how the mutation contributed to disease.

More recent work has shown that the mutation mucks up the machinery that the cell uses to turn DNA into RNA, and then into protein. Instead of beginning at the traditional specific “start” line, the large number of repeats causes the translational machinery to start at a variety of locations on the repeat RNAs. This creates a variety of unusual toxic proteins made up of two amino acids, which have been found in cell culture, animal models, and patients with ALS. That these dipeptide repeat (DPR) proteins were toxic was clear, but researchers still weren’t sure precisely how they were harming the cell.

Fen-Biao Gao is a Packard Center researcher and a neuroscientist at the University of Massachusetts Medical School.

Packard scientist Fen-Biao Gao and his postdoc Helene Tran started to create an animal model of C9orf72 as soon as they heard about the mutation’s discovery. They started this work in the fruit fly Drosophila melanogaster, inserting the repeat expansion linked to ALS in humans into the fruit fly genome. Gao and Tran didn’t insert the DNA just anywhere, however. In humans, the C9orf72 mutation is in one of the gene’s introns, which means it is removed during the journey from DNA to protein and doesn’t normally get translated. So Gao and his colleagues made sure to put the long string of repeated nucleotides in a human intron to replicate human biology as closely as possible.

At first, their intron model appeared to be a flop. The flies were almost appallingly healthy and normal without significant changes in gene expression, even though small aggregates of sense RNA (that is, RNA that was transcribed in the forward direction) appeared in the nuclei of flies with 160 repeats 10 times more abundantly than in human neurons. They didn’t show any signs of motor neuron disease. In the meantime, other scientists had placed the repeat expansion directly in the context of a messenger RNA with polyA tail, which meant the mutation was easily turned into toxic DPR proteins. Gao and Tran confirmed that these fruit fly models of C9orf72 were exceedingly lethal and produced more than 100 times more DPR proteins than the intron model. However, these flies did not have nuclear RNA foci because repeat RNAs were actively transported to the cytosol. Gao’s intron model started to create doubt that the sense RNA foci in the nucleus were toxic. If they were, then Gao and Tran’s flies should also be showing signs of damage, but they weren’t.

Gao and Tran returned to the intron model and turned up the heat in the flies—literally. Instead of raising the fruit flies at 25° Celsius, they cranked up the thermostat four degrees. At 29° Celsius, the flies began dying slightly younger than those raised at a cooler temperature. But when Gao and Tran checked, they found that flies at both temperatures had similar numbers of sense RNA foci. What was different between both groups of insects, however, was the presence of dipeptide repeats. In particular, they found four times more of a particular dipeptide repeat protein known as poly-glycine-proline in the flies raised at a more balmy 29° Celsius. These seemed to suggest that what was harming people with the C9orf72 mutation were not sense RNA foci, but the DPR proteins made in the cytosol.

Gao said that he expected his results to be controversial, since many researchers have focused on RNA foci as being toxic by sequestering important RNA-binding proteins. However, it is not the case at least in this animal model. He cautioned that it remains possible some other forms of repeat RNAs, such as nuclear antisense RNA foci, might be able to sequester a sufficient quantity of specific proteins and be responsible for some aspects of neurodegeneration.

Even outside of these specific findings, Gao thinks the Drosophila intron model he and Tran developed will be useful in understanding other aspects of ALS. “Because the repeats are in the human intron, it’s similar to what’s actually going on biologically in humans. Thus, the intron model will be useful to figure out how expanded repeats expressed in their native molecular context can eventually compromise motor neurons under certain circumstances,” Gao said.